Generation Of A Monoclonal Antibody Against Streptococcus Suis Serotype 2 SSU05-0474 Protein And Characterization Of Its Epitopes

Streptococcus suis serotype 2 (SS2) is a zoonotic infectious disease pathogens. SS2 infection occurs in many countries around the world, causing meningitis septicemia, arthritis, endocarditis, poisoning shock syndrome both in human and animals, resulting in huge economic loss and seriously threating human health.In order to prevent and control the infection of SS2, researchers have been committed to the study of its pathogenesis and have found many SS2 virulence factors, including capsular polysaccharides (CPS), muramidase-released protein(MRP), suilysin (SLY), extracellular factor (EF)and LPXTG protein. The last one directly contacts with the host cells or proteins, and play important roles in pathogenicity. According the genomic sequence analysis, we found an LPXTG protein from genome of SS2 05ZYH33 strain (SSU05-0474). The protein encoded by this gene has a high homology with the major pili subunit protein, so in this paper it was named SsMps. Our preliminary study showed that SsMps has the ability of binding to an important host immune factor, indicating it may play a role in the pathogenesis of SS2.In this study, SSU05-0474 gene was PCR-amplified using the genomic DNA of SS2 strain 05ZYH33 as template and cloned to pET22b expression vector to construct recombinant plasmid pET22b/ssMps. The recombinant plasmid was sequenced and then was transformed into E. coli BL21 (DE3). SsMps protein was successfully induced to a high expression level with final concentration of 0.8 mmol/L IPTG under 22℃ overnight. Western-Blot confirmed that the recombinant protein SsMps with His-tag was successfully expressed.High purity of SsMps protein was purified by affinity chromatography. Recombinant protein with molecular weight of 45 kDa was analyzed and confirmed by SDS-PAGE.The recombinant protein SsMps was used as antigen to immune BALB/C mice for three times. One monoclonal antibody named 1D9 was obtained by cell fusion and screening. Western-blot showed 1D9 specially recognized the SsMps protein in SS2 cell fraction. Subtype assay indicated that 1D9 was IgG1 with the κ light chain. This monoclonal antibody can be used for further SsMps protein function study.To identify the antigen epitope of the monoclonal antibody 1D9, phage random peptide library was screened for 3 rounds, and 18 phage clones which showed positive binding to 1D9 were obtained. Sequencing results revealed that 7 phage clones had consensus sequences LPQRRQTRIMII, and 2 had RRHTTRKRMLKT, sharing similarity with amino acid sequence 162-183 and 445-466 of SS2 SsMps protein, respectively. The corresponding synthetic peptides competitively inhibited binding between monoclonal antibodies and SsMps, indicating the phage displayed epitopes were similar with the natural epitopes of SsMps protein in S. suis serotype 2. Thus, it was presumed that the epitope of 1D9 fall within the range of these two sequences...